Fig. 2

EAE induces lymphangiogenesis near the cribriform plate. a, b Nine representative serial coronal sections of the lymphatic vessels near the cribriform plate were imaged from either a healthy (a) or an EAE Score 3.0 collected 18 days post-immunization (b) CD11c-eYFP transgenic reporter mouse were immunolabeled with Lyve-1 and DAPI. Note the increase in Lyve-1⁺ vessel area during EAE versus healthy mice. Scale bars = 100 μm. c–h Enlarged images of the red inset in a or yellow inset in b showing the section with the largest Lyve-1⁺ vessel area in healthy (c–e) versus EAE Score 3.0 (f–h) out of nine serial coronal sections. Scale bars = 100 μm. i, j Quantitation of the average Lyve-1⁺ vessel area across nine serial sections (i) (n = 4 mice per group; data are represented as mean ± SEM, ***p < 0.001, repeated two-way ANOVA using Sidaks multiple comparison test) or the average Lyve-1⁺ vessel volume (j) (n = 4 mice per group; data are represented as mean ± SEM, *p < 0.05, unpaired Student’s t-test). k–t Proliferation of lymphatic endothelial cells measured by Ki67⁺ nuclei within healthy (k–o) or EAE Score 3.0 mice (p–t). Ki67⁺ Lyve-1⁺ lymphatic endothelial cells were quantified using orthogonal views to confirm that the cells used for quantitation were in fact proliferating lymphatic endothelial cells. Yellow arrowheads indicate Ki67⁺ nuclei within Lyve-1⁺ vessels. Scale bars, 100 μm in a–t. Scale bars = 100 μm. u Quantitation of the average percent of Ki67⁺ nuclei within Lyve-1⁺ vessels between healthy and EAE Score 3.0 mice (n = 4 mice per group; data are represented as mean ± SEM, *p < 0.05, unpaired Student’s t-test